PROJECT SUMMARY/ABSTRACT: The ability to genetically modify model organisms to study gene function has undoubtedly been an enormous benefit to biomedical science as a whole and has significantly advanced what scientists and clinicians can achieve to further scientific knowledge and improve patient outcomes. However, the ability to correct genetic defects or to induce precise changes to the DNA of living organisms is still limited. Precise regulation of nucleotide changes in specific cell populations of living organisms, including humans, would greatly advance scientific knowledge. In particular, the field of behavioral neuroscience would greatly benefit by having a set of tools that could reliably manipulate gene function precisely, at specific time points during behavioral analysis. For example, the molecular basis of many behavioral and psychological phenomena, such as the mechanisms that govern learning and memory, could be parsed out faster and more efficiently. The recent development of a genome editing tool, which is based on the RNA-guided Cas9 nuclease from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has brought us closer to reaching this goal. The CRISPR/Cas9 system has been shown to be capable of inactivating genes using the error prone non-homologous end joining (NHEJ) DNA repair mechanism in neurons in vitro and most recently in vivo. However, despite this recent progress, there are still many unanswered questions regarding the efficiency and fidelity of the CRISPR/Cas9 system for genome editing within the intact brain. There simply has not been enough unbiased evaluation of the fidelity of in vivo genome editing within the brain to warrant its use in behavioral neuroscience. Our proposed experiments will close this knowledge gap and position our study to be the most comprehensive unbiased analysis of in vivo off target editing to date. Additionally, researchers are currently limited in their ability to induce genome editing in a wide range of cells, in a cell type-specific manner with precise temporal control. This proposal will improve this technology by developing the means to create specific modifications of the genome at precise loci in discrete populations of cells in the brain of living mammals. We will develop a viral delivery system that can deliver the critical components of the CRISPR/Cas9 system to the mammalian brain, allowing specific cell types to be targeted for genome editing in an inducible manner where the genome editing system can be induced using doxycycline. We will also generate a Cre recombinase-dependent genome editing system that can be utilized on its own, or in conjunction with the inducible system we will generate. This technique will provide an unprecedented ability for spatial and temporal control of genome editing, and allow genome editing to occur in specific cell types in vivo at any point during behavioral analysis.